Time of flight mass spectrometry from gel isolates or chromatographic columns are accepted means of identifying proteins based on mass and charge (m/z). In 1988, Koichi Tanaka reported that laser irradiation of a mixture of methanol-ethylene glycol-cobalt ultra fine powder (Co UFT) transferred energy to proteins, chymotrypsinogen and lysozyme, generating a vapor/ion-phase of intact macro molecules that could be detected by time of flight (TOF) mass spectrometry (Tanaka, et al., Rapid Commun. Mass Spectrom. 1988, 2, 151-153). This demonstration paved the way for using laser desorption ionization mass spectrometry on solid tissue, direct tissue MALDI (DT-MALDI).
MALDI (TOF) identifies proteins and peptides as mass charge (m/z) spectral peaks. Further advances, post-source decay (MALDI-PSD) and collision-induced dissociation (MALDI-CID), have become standard methods to identify proteins from cell or tissue homogenates that are responsible for these peaks. (Hansen, et al., Molecular & Cellular Proteomics 2003, 2, 299-314; Norris, et al. Anal Chem. 2003, 75, 6642-6647; Zhang, et al., Am Soc Mass Spectrom 2003, 14, 1012-1021; Wang, et al. Journal of Proteome Research 2005, 4, 2397-2403; Vasilescu, et al., Journal of Proteome Research 2005, 4, 2192-2200; Vandermoere, et al., Oncogene 2005, 24, 5482-5491; Luo, et al., Molecular Biotechnology 2005, 29, 233-244; Le Guezennec, et al., Molecular and Cellular Biology 2006, 26, 843-851. However, most of these procedures utilize harsh digestion conditions and protein extraction procedures prior to analysis by MALDI-TOF methods. Matrices have evolved to include small organic molecules and heavy metals that that can be applied to or mixed with the analyte (Tanaka, et al., Rapid Commun. Mass Spectrom. 1988, 2, 151-153). The most often used matrices absorb light at 337λ, the wavelength of a nitrogen laser, and thereby facilitate desorption and ionization of adjacent biological materials. Ions of the same charge acquire a similar kinetic energy; however, their velocity in the ion chamber depends on their respective masses. The ion time of travel to an anode is measured, precisely by the detector and is recorded as a time-mass/charge spectrum with peaks representing proteins in the sample. Based on instrument calibration of standard samples, these values are converted to mass values for National Center for Biotechnology Information (NCBI) database analysis of peptide fragments with Mascot©, and Blast© software. Protein m/z Spectra can be obtained from fluids, cells and tissues of matrix coated protein extraction preparations with Maldi tof and Maldi tof tof. Although protein signature peaks are reported (Norris, et al., Analytical Chemistry 2003, 75, 6642-6647), there have been no reports of proteins identified directly from cells or tissue using these technologies. Thus, there is a need in the art to provide a more rapid, yet accurate method to identify proteins directly from a tissue sample without a need for further protein extraction of the sample to be followed by protein determination using standard methods such as 1D, 2D or capillary electrophoresis. These needs are addressed by the agents and methods of the present invention.
All publications, patent applications, patents and other reference material mentioned are incorporated by reference in their entirety. In addition, the materials, methods and examples are only illustrative and are not intended to be limiting. The citation of references herein shall not be construed as an admission that such is prior art to the present invention.